Background It remains unclear whether serum total cholesterol is associated with colorectal cancer (CRC) risk. Interplay between dietary fatty acids and serum total cholesterol on CRC risk may be present as well. We aimed to investigate the association between serum total cholesterol with CRC. Furthermore, we investigated whether this association was modified by intake of dietary polyunsaturated fatty acids (PUFAs). Methods We analysed data from 6628 participants of the Rotterdam Study, a prospective population-based follow-up study among patients aged 55years and older. Serum total cholesterol was measured at baseline. During a mean follow-up time of 12.9years, we identified 248 new CRC cases based on pathology data and medical records. Multivariable HRs were calculated using Cox regression models. Results After adjustment, serum total cholesterol levels were associated with a higher risk of CRC (HR 1.49; 95% CI 1.08 to 2.06 for highest vs lowest tertile). Statistically significant effect modification was present for PUFAs intake (P-interaction=0.04). After stratification by median PUFAs intake, an increased risk with increasing tertiles of serum total cholesterol was observed among patients with low PUFAs intake (3rd tertile vs 1st tertile: HR 2.43; 95% CI 1.41 to 4.18), whereas no association was observed among patients with high PUFAs intake (3rd tertile vs 1st tertile: HR 0.93; 95% CI 0.55 to 1.58). Conclusions Taken together, these findings suggest that high levels of serum total cholesterol increase CRC risk, but this risk may be reduced by high dietary PUFAs intake.